Roche Diagnostics Operations, Inc.

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Christian Rudolph

Harald Sobek

Johann-Peter Thalhofer

Michael Greif

Manfred Schmidt

Rainer Mueller

Armin Ruf

The present disclosure provide novel variants of T7 RNA polymerase. Embodiments of T7 variants, according to the instant invention, include a Cysteine-Serine substitution on position 723 of the amino acid sequence of the T7 polypeptide. Embodiments of T7 variants according to the instant invention have a DNA-dependent RNA polymerase enzymatic activity and a reduced tendency to form intramolecular homodimers by way of oxidizing thiol groups. The amino acid substitutions within the T7 variants disclosed herein impact minimally, if at all, the RNA polymerase activity of the T7 polypeptide. Further, the mutations of the disclosed embodiments may optionally be combined with mutations which provide enhanced thermostability compared to the wild-type reference.

T7 RNA polymerase variants with cysteine-serine substitutions

The present invention provides improved variants of T7 RNA polymerase by introducing novel mutations which lead to improved thermostability of the enzyme. According to the invention, amino acid substitutions at the positions Val426, Ser633, Val650, Thr654, Ala702, Val795, and combinations thereof are advantageous.

T7 RNA polymerase variants with enhanced thermostability

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Penzberg, Germany

Christian Rudolph